Journal of Chromatography A, 1129 (2006) 300303

Short communication

Development and single-laboratory validation of a new gas

chromatographic multi-pesticide method of analysis of commercial
emulsifiable concentrate formulations containing alachlor, chlorpyrifos
methyl, fenthion and trifluralin as active ingredients
Helen Karasali a, , George Balayannis a , Adamantia Hourdakis a , Arpad Ambrus b,1
a
Benaki Phytopathological Institute, 8 St. Delta Street, Kisia 14561, Athens, Greece
b Arpad Ambrus, FAO/IAEA Agriculture and Biotechnology Laboratory, Wagramerstrasse 5, Vienna, Austria
Received 16 December 2005; received in revised form 8 August 2006; accepted 14 August 2006

Abstract
A multi-pesticide (MP) method was developed and single-laboratory validated for the quality control of commercial pesticide products containing
alachlor, chlorpyrifos methyl, fenthion and trifluralin as active ingredients (a.i.). A capillary gas chromatographic system with flame ionization
detection (FID) and a programmable temperature vaporising split injector was used. The performance characteristics (specificity, linearity, precision
and repeatability) of the method satisfied international acceptability criteria.
2006 Elsevier B.V. All rights reserved.

Keywords: Multi-pesticide; Method validation; Alachlor; Chlorpyrifos methyl; Fenthion; Trifluralin

1. Introduction
Properly used, pesticides are essential for the provision of
food requirements for the worlds ever growing population. Ben-
efits consist not only in increased production yields, but also in
better quality. For successful pest control, the use of pesticide
products of known quality is essential. For that reason, almost
all countries have regulatory authorities that monitor the quality
of pesticide products. The recommended methods are collabora-
tively tested and published by Association of Official Analytical
Chemists (AOAC) and Collaborative International Pesticides
Analytical Council (CIPAC). Usually, an analytical method pre-
sented for collaborative trial through AOAC or CIPAC is a
method developed by a manufacturing company. Hence, these
methods are valid only for particular formulations prepared by
specific manufacturers. These methods are optimised for those
specific products and conditions. Each chromatographic method
Corresponding author. Tel.: +30 210 8077587; fax: +30 210 8077506.
E-mail addresses: chem3@bpi.gr (H. Karasali), A.Ambrus@iaea.org
(A. Ambrus).
1 Tel.: +43 1 260028395; fax: +43 1 2600 28222.
has its own stationary phase, internal standard (I.S.) and mobile
phase. The rapid expansion in the use of chromatographic meth-
ods for pesticide products analysis has led to a wide choice of
chromatographic columns and variation in experimental param-
eters, along with increased demands on instrumentation. Whilst
this approach suits agrochemical manufacturers and suppliers
of a limited range of specific products, its inherent complex-
ity can cause problems when implemented on a global basis in
laboratories where many different pesticide products have to be
analysed. However, due to the great variety of active ingredients
(a.i.) and formulations of pesticides to be monitored, the need
for new methods with higher sample output and lower cost of
analysis has become imperative. An answer to this need is the
development of multi-pesticide (MP) methods.
Although a great number of multi-residue methods have been
developed [13] for the determination of pesticide residues
in various matrices, surprisingly enough, the idea for multi-
pesticide methods has only recently started to develop. This
explains the very small number of publications for MP meth-
ods, which can be found in international literature [4,5].
A multi-pesticide method is one that determines the active
ingredients content of each of a range of commercial pesticide
formulations, using the same chromatographic column and elu-

0021-9673/$ see front matter 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.chroma.2006.08.040
 H. Karasali et al. / J. Chromatogr. A 1129 (2006) 300303
tion system. The test portions must be prepared and extracted
according to a collaboratively tested CIPAC or AOAC method,
if available, in order to assure comparable results with the stan-
dard procedures. In the case of multi-pesticide methods samples
are analysed separately from each other. In one sample, no more
than two known active ingredients are usually to be separated
from the impurities of the technical material and components of
formulations. In contrast, in the multi-residue analysis, a lot of
active ingredients are analysed [4] simultaneously.
In the case of MP methods, instrumental determination is car-
ried out with a notably limited number of properly selected gas
chromatography (GC) or high-performance liquid chromatogra-
phy (HPLC) column/elution systems, in contrast to the current
AOAC and CIPAC methods.
The reduced number of columns and elution systems
required, in combination with the reduction of running costs,
contribute to a significant decrease in the cost of analysis for an
important variety of pesticides.
The present study, which is part of the FAO/IAEA Quality
Control of Pesticides project (whose aim is to incorporate
a great number of pesticide compounds to the method in the
future), is about the development and validation of a multi-
pesticide gas chromatographic method [GC-flame ionization
detection (FID)] for the quantitative determination of four active
ingredients (alachlor, chlorpyrifos methyl, fenthion and triflu-
ralin) in their commercially available emulsifiable concentrate
(EC) formulations, which are widely used in Greece. These
four active ingredients were selected for the initial development
of the method. Although the above-mentioned pesticides have
been on the market for many years, the development and
validation of a new analytical method is almost imperative. In
the case of alachlor [6], the CIPAC official method could be
considered outdated, since it involves the use of a GC-glass
packed column, whereas the method for trifluralin is not well
specified [7]. In the case of fenthion the method is a spectropho-
tometric one [8] and there is no CIPAC method for chlorpyrifos
methyl.
2. Experimental
2.1. Materials
The analytical standards of trifluralin (99.4%) and chlorpyri-
fos methyl (99.8%) were donated from Dow Agrosciences (Nor-
folk, UK); of fenthion (96.2%) from Bayer (Athens, Greece);
and of alachlor (99.8%) from Monsanto (Antwerp, Belgium).
Dipentyl phthalate (99% pure, purchased from Noechema,
Bodenheim, Germany) was used as internal standard. Each of
the analytical standards as well as the internal standard was
supplied with a certificate of analysis (CoA) stating their concen-
tration, determined by the supplier. For each active ingredient
five different batches of commercially available EC formula-
tions together with their blank formulations were given by the
suppliers. These batches, accompanied by their CoA, and were:
Treflan 480EC (trifluralin 48%, w/v), Reldan 2E (chlorpyrifos
methyl 22%, w/v), Lebaycid 500EC (fenthion 50%, w/v) and
Lasso EC (alachlor 48%, w/v).
301
2.2. Individual stock and working solutions
An internal standard solution of dipentyl phthalate
0.1512 mg ml1 was prepared in acetone (pesticide residue
grade). Stock solutions for each active ingredient were prepared
by diluting the appropriate amount of the respective analytical
standard with the internal standard solution at concentrations
of about 0.8 mg ml1 . Three working solutions for each a.i.
(low calibrated level, medium calibrated level, and high cali-
brated level) were prepared by three independent dilutions of
the stock solution with internal standard solution at concentra-
tions of about 0.8, 1.0 and 1.2 times the nominal concentration
of a.i. in the formulated product, respectively.
2.3. Sample preparation
For the preparation of the sample solution, the appropriate
quantity of the commercial EC formulation, containing approx-
imately 80 mg (5%) of active ingredient, was diluted with the
internal standard solution (concentrated sample extracts). Suffi-
cient quantity of this solution was diluted with internal standard
solution, so that the final concentration fell within the concen-
tration range of the respective working solution (diluted sample
extracts).
For each active ingredient, a concentrated blank solution was
prepared by diluting a quantity of the blank formulation with ace-
tone, followed by evaporation of the solvent to half the volume.
The concentrated blank solutions were used for the evaluation
of the specificity of the method.
2.4. GC System and conditions
The gas chromatographic system used was a ThermoFinni-
gan Trace GC instrument (ThermoFinnigan Italia, Rodano,
Italy), consisting of a programmable temperature vaporiz-
ing (PTV) split injector, a FID system and an autosampler
(ThermoFinnigan AS 2000). Two columns of different polarity
were used: a low polar CP-Sil 8Cb (Varian), 25 m 0.53 mm
I.D., 1 m film thickness and a medium polar DB 1701
(Agilent Technologies, USA), 15 m 0.53 mm I.D., 1 m film
thickness.
The operating conditions for CP-Sil 8Cb were: helium as car-
rier gas set at pressure 0.043 MPa, split flow set at 95 ml min1 ,
split ratio set at 13. The temperature program was the following:
the oven temperature was held at 80 C for 1 min, then heated
to 200 C at a heating rate of 35 C min1 , and kept at that tem-
perature for 19 min.
The operating conditions for DB-1701 were: helium as car-
rier gas set at pressure 0.013 MPa, split flow set at 45 ml min1 ,
split ratio set at 13. The temperature program was the following:
the oven temperature was held at 80 C for 1 min, then heated
to 220 C at a heating rate of 35 C min1 , and kept at that tem-
perature for 10 min.
The carrier gas was ultra-high-purity helium (99.9999%,
AirLiquide, France).
In both cases the detection temperature was set at 250 C and
the injection volume at 0.5 l.
302 H. Karasali et al. / J. Chromatogr. A 1129 (2006) 300303

The temperature program of the PTV split injector for both columns was:
Injection phase 60 C for 0.05 min
Evaporation phase From 60 to 150 C at a heating rate of 14.5 C s1 , remained at 150 C for 0.5 min
Transfer phase From 150 to 250 C at a heating rate of 14.5 C s1 , remained at 250 C for 0.5 min
Cleaning phase 1 min

3. Elaboration of a multi-pesticide method
Before the method development, the system suitability test
of the chromatographic system had to be performed. The system
suitability test assures good column performance and stability
of instruments, which means good quality of results [9]. The
procedure for the elaboration of the multi-pesticide method used
was:
(1) Determination of the initial optimum chromatographic con-
ditions for the selected pesticides.
(2) Repeatability testing of GC injection.
(3) Specificity test. Extraction of blank formulations without
internal standard and analysis of these extracts under the
selected chromatographic conditions.
(4) Sample and working solutions preparation.
(5) Evaluation of linearity of response. Range. Multi-point cal-
ibration (3 2 points).
(6) Determination of critical parameters of calibration (linear-
ity, correlation coefficient, slope and intercept with confi-
dence intervals, standard deviation of relative residuals).
(7) Determination of method precision. Sample analysis.
(8) Repetition of the above stages to a second column of differ-
ent polarity.
(9) Comparison of the results and evaluation of the method.
4. Results and discussion
Both CIPAC [10] and EU [11] guidelines recognize that it
is necessary to evaluate the following analytical performance
parameters.
4.1. Repeatability of injections
The repeatability of injections was tested for each active
ingredient separately, using the medium calibrated level work-
ing standard solution, following the selected chromatographic
conditions. In case the relative standard deviation (RSD) of the
peaks area ratios (a.i./I.S.) is 1%, the repeatability test is con-
sidered acceptable, as it was in our case.
4.2. Specicity
The ability of the analytical method to distinguish the ana-
lyte to be determined from degradation products, metabolites or
known additives was investigated [10,11].
Lack of interference is demonstrated by the analysis of
the concentrated blank formulations and concentrated sample
extracts. The use of a second column of different polarity also
confirmed interference-free separation.
4.3. Linearity of response. Range
The linearity of response was determined by analyzing in
duplicate three working solutions of different concentrations for
each of the tested active ingredients [10,11] (Table 1). The range
for each active ingredient is presented in Table 1. Linearity is
acceptable when the correlation coefficient is 0.997 [10,11]
and the standard deviation of relative residuals is 0.01. After
having performed the multi-point calibration (3 2-injections),
correlation coefficient (r), slope and intercept, with confidence
limits at 95% level, were determined using analysis of variance
(ANOVA). The standard deviation of relative residuals (sYrel )
was also determined (Table 1). Similar results were obtained
with column DB 1701.
4.4. Precision of method. Sample analysis
Data obtained from the analysis in duplicate of samples from
five different batches of each pesticide was used to calculate
the experimental RSDr values. The acceptability of the results
should be based on the modified Horwitz equation (Eq. (1))
[1012].
RSDr (%) = RSDR (%) 0.67 (1)

Table 1
Linearity of response
Active ingredient Level of calibration Slope a SDa Intercept b SDb r sYrel Concentration range (mg ml1 )

Alachlor 6 0.8813 0.0030 0.026 0.006 1.000 0.0016 0.20140.4030

Chlorpyrifos methyl 6 0.3497 0.0040 0.015 0.009 0.9998 0.0058 0.24740.4109
Fenthion 6 0.6821 0.0045 0.0036 0.01 0.9999 0.004 0.20630.4106
Trifluralin 6 0.6679 0.0038 0.006 0.0088 0.9999 0.0039 0.19660.4645

Slope and intercept with confidence limits, correlation coefficient (r), relative residuals (sYrel ) and concentration range for alachlor, chlorpyrifos methyl, fenthion
and trifluralin on column CP Sil 8 Cb.
 H. Karasali et al. / J. Chromatogr. A 1129 (2006) 300303 303

Table 2
Comparison of the results of Lebaycid 50EC (fenthion), Lasso 48EC (alachlor), Reldan 2E (chlorpyrifos methyl), Treflan 48EC (trifluralin), obtained with two
different columns, with the paired t-test
Sample no. Fenthion Alachlor Chlorpyrifos methyl Trifluralin

Difference of the averages Difference of the averages Difference of the averages Difference of the averages

1 0.011 0.024 0.027 0.066

2 0.009 0.016 0.010 0.071
3 0.008 0.002 0.012 0.065
4 0.005 0.004 0.010 0.078
5 0.006 0.010 0.013 0.060
Average 0.008 0.011 0.014 0.068
SDdif 0.002 0.009 0.007 0.007
RSD 0.306 0.805 0.497 0.100
tcalc = 0.684 1.799 1.112 0.224
tcrit = 2.776 2.776 2.776 2.776

where RSDr is the repeatability relative standard deviation and Acknowledgement

RSDR is the inter-laboratory relative standard deviation.
This study has been supported by the International Atomic
4.5. Comparison of the results obtained with two different Energy Agency (IAEA), Co-ordinated Research Project Qual-
columns ity Control of Pesticides (contract No. 11772/RO).

The results obtained from the analysis in duplicate of the References

samples from five different batches of each pesticide, with the
two different columns were compared with the paired t-test.
It can be concluded that the results obtained with the two
columns were not significantly different (Table 2), as long as
tcalc was tcrit for all active ingredients.
So, the MP method, consisting of chromatographic analysis
on two columns, is validated for the tested pesticides.
Based on the performance parameters obtained during the
multi-pesticide method validation, we can conclude that the
method is suitable for determining the four selected pesticides
with the same chromatographic system.
The method proved to be more convenient than the official
methods for routine analysis of the pesticide formulations. The
operation cost as well as the total time of analysis is signifi-
cantly reduced. As far as the chromatographic CIPAC methods
are concerned [68], different packed (outdated) columns, dif-
ferent carrier gases with high flow rates, different temperature
programs and different internal standards must be used. This
results in increased operation cost, inconvenience for the analy-
sis of different pesticides and time consuming procedures.
The development of MP methods is a challenging field of
research as long as its results can be readily applied for the
cost-effective determination of the active ingredients of various
pesticides.
The use of the multi-pesticide method is a practical alternative
for the analysis of pesticides, of which the official methods are
formulation-specific.
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